Studies of the hormone-binding protein neurophysin are proposed that will build on developments from the past project period, which include elucidation of the crystal structure of a neurophysin-peptide complex, to address fundamental questions about protein folding mechanisms, allosteric interactions and molecular recognition. These studies should ultimately be useful also in understanding relationships between defects in neurophysin structure and disorders of vasopressin synthesis. Neurophysins and their associated peptide hormones share common biological precursors that are processed to give the mature protein and hormone. The proposed studies of neurophysin folding pathways are based on the observation that the disulfides of unliganded neurophysin are metastable and that reduced neurophysin folds efficiently only in the presence of ligand peptides, conditions that mimic folding of the precursor. These studies are aimed at identifying the stage in folding at which the peptide exerts its influence, and details of the mechanism by which it acts. The relative folding properties of the two homologous neurophysin domains will also be investigated to test the hypothesis that the principal folding defect in the absence of peptide arises from the instability of the 10-54 disulfide bridge, which is located directly at the hormone-binding site and which is unique to the amino domain. The binding of peptides to the hormone-binding site of neurophysin is accompanied by a large increase in neurophysin dimerization constant, and also by a conformational change within the dimer that may contribute significantly to the unusual thermodynamics of the reaction. Because only the conformation of the liganded dimer is available from the crystal structure, NMR studies of the relative conformations of liganded and unliganded dimers, and of unliganded monomers and dimers, are proposed with the aim of elucidating the structural basis of the ligand-facilitated dimerization and the nature of ligand-induced changes in secondary and tertiary structure. The hormone-binding site of neurophysin binds the aromatic ring in position 2 of ligand peptides with very high specificity and affinity. Molecular simulations of the binding pocket, based on the crystal structure, will be carried out to elucidate the structural origins of this property. In other areas, studies of the neurophysin precursor are proposed in order to: a) thermodynamically compare hormone-neurophysin interactions within the precursor with those involved in complex formation, as an approach to the evaluation of entropic factors involved in these reactions, and b) study folding pathways within the precursor itself. Both chemical and molecular biological routes to obtain precursor are proposed. Studies of the role of the copeptin segment of the vasopressin- and related precursors are proposed, using an ostrich neurophysin to which this segment remains attached in the isolated protein.